This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The dramatic progress in high throughput proteomics analysis via ESI-LC/MS over the last decade has been fueled to a large degree by continuous improvements in instrumentation. High throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become the dominant analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ VelosTM) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times two times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. Faster cycle times on the new instrument alternatively allow for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. The greatly increased ion flux into the mass spectrometer also results in greatly increased detection of low abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker?s yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Additionally, mass analysis in the new instrument can be carried out with resolution in excess of 20,000 FWHM, sufficient for isotopic resolution and top down experiments of small intact proteins, such as myoglobin.